Activated protein C (APC) has been approved to treat patients with sepsis but the mechanism for APC's protective effects in systemic inflammation is not understood. Breakdown of the barrier that separates the blood from tissues plays a key role in inflammatory disorders. This proposal is based on our discovery that protease activated receptor-1 (PAR1)-dependent signaling by exogenous and endogenously generated APC strongly enhances endothelial barrier integrity in cell culture. PAR1 is efficiently activated by thrombin and it is currently not understood how APC-PAR1 signaling can be relevant under conditions where thrombin is also present, such as in sepsis. In Aim 1 we will investigate whether thrombin- but not APC- activated PAR1 is efficiently internalized from the endothelial cell surface because thrombin remains bound to PAR1. We will test whether blocking thrombin binding to PAR1 leads to a conformation of thrombin-cleaved PAR1 that resembles APC-cleaved PAR1 and supports protective downstream signaling. Aim#2 will test the hypothesis that PAR1 signaling by the PC pathway in endothelial cells reduces adhesion and transendothelial migration of neutrophils and monocytes. We will analyze expression of endothelial adhesion receptors and leukocyte adhesion and transmigration under static conditions and in parallel plate hydrodynamic flow chambers. Effects of PC pathway signaling on rolling, adhesion, and transmigration of neutrophils and monocytes will be established. Aim#3 is to define the in vivo effects of PC pathway signaling on endothelial barrier properties in mouse models. We will characterize PC activation in vivo in response to human and murine thrombin variants. We will establish whether PAR1 signaling by the PC pathway can reduce hyperpermeability of the vascular barrier in response to injection of vascular endothelial growth factor and downregulate expression of adhesion receptors. The effect of PC plasma levels on APC signaling will be evaluated. The response to exogenous and endogenously generated APC will be determined in different tissues to establish how efficiently the PC pathway links to protective signaling in specific vascular beds. These three interrelated aims will provide conceptually novel insight into how protective PAR1 signaling by the PC pathway prevents the spreading of barrier dysfunction upon injury. The results will have important implications for future treatment strategies in patients. PUBLIC HEALTH RELEVANCE: The coagulation factor APC has been approved to treat patients with sepsis. In order to understand how the powerful anti-inflammatory effects of APC are mediated, we will analyze how APC affects properties of the vascular barrier that separates the blood from underlying tissues. The results will have important implications for novel treatment strategies for diseases where the inflammatory response plays a key role, including myocardial infarction and stroke.